Tài liệu Báo cáo khoa học: NMR structure of AII in solution compared with the X-ray structure of AII bound to the mAb Fab131 pptx

On the molecular basis of the recognition of angiotensin II (AII)
NMR structure of AII in solution compared with the X-ray structure of AII bound
to the mAb Fab131
Andreas G. Tzakos
1
, Alexandre M. J. J. Bonvin
2
, Anasstasios Troganis
3
, Paul Cordopatis
4
, Mario L. Amzel
5
,
Ioannis P. Gerothanassis
1
and Nico A. J. van Nuland
2
1
Department of Chemistry, Section of Organic Chemistry and Biochemistry, University of Ioannina, GR-45110, Greece,
2
Bijvoet Center for Biomolecular Research, Department of NMR Spectroscopy, Utrecht, the Netherlands;
3
Department of Biological
Applications and Technologies, University of Ioannina, Greece;
4
Department of Pharmacy, University of Patras, Greece;
5
Department of Biophysics and Biophysical Chemistry, Johns Hopkins University, School of Medicine, Baltimore, MD 21205, USA
The high-resolution 3D structure of the octapeptide hor-
mone angiotensin II (AII) in aqueous solution has been

obtained by simulated annealing calculations, using high-
resolution NMR-derived restraints. After final refinement in
explicit water, a family of 13 structures was obtained with a
backbone RMSD of 0.73 ± 0.23 A
˚
. AII adopts a fairly
compact folded structure, with its C-terminus and N-ter-
minus approaching to within  7.2 A
˚
of each other. The side
chains of Arg2, Tyr4, Ile5 and His6 are oriented on one side
of a plane defined by the peptide backbone, and the Val3 and
Pro7 are pointing in opposite directions. The stabilization of
the folded conformation can be explained by the stacking of
the Val3 side chain with the Pro7 ring and by a hydrophobic
cluster formed by the Tyr4, Ile5 and His6 side chains.
Comparison between the NMR-derived structure of AII in
aqueous solution and the refined crystal structure of the
complex of AII with a high-affinity mAb (Fab131) [Garcia,
K.C., Ronco, P.M., Verroust, P.J., Brunger, A.T., Amzel,
L.M. (1992) Science 257, 502–507] provides important
quantitative information on two common structural fea-
tures: (a) a U-shaped structure of the Tyr4-Ile5-His6-Pro7
sequence, which is the most immunogenic epitope of the
peptide, with the Asp1 side chain oriented towards the
interior of the turn approaching the C-terminus; (b) an Asx-
turn-like motif with the side chain aspartate carboxyl group
hydrogen-bonded to the main chain NH group of Arg2. It
can be concluded that small rearrangements of the epitope
4–7 in the solution structure of AII are required by a mean

value of 0.76 ± 0.03 A
˚
for structure alignment and
 1.27 ± 0.02 A
˚
for sequence alignment with the X-ray
structure of AII bound to the mAb Fab131. These data are
interpreted in terms of a biological ÔnucleusÕ conformation of
the hormone in solution, which requires a limited number of
structural rearrangements for receptor–antigen recognition
and binding.
Keywords: angiotensin II; monoclonal antibody; NMR;
peptide structure; VIb turn.
Angiotensin II (AII), the main effector octapeptide hor-
mone (Asp1-Arg2-Val3-Tyr4-Ile5-His6-Pro7-Phe8) of the
renin–angiotesin system [1], exerts a variety of actions on
different target organs via specific receptors designated AT
1
and AT
2
[2,3]. Most of the known physiological effects of
AII have been attributed to AT
1
, e.g. vasoconstriction,
aldosterone release, renal sodium reabsorption, as well as
central osmoregulatory actions, including the release of
pituitary hormones into the circulation and growth stimu-
lation in various cell types. These effects constitute the role of
angiotensin peptides as neuromodulators/neurotransmitters
in the brain. Because of the variety of biological and

physiological actions of AII in various tissues, intensive
research is required to determine the structural features of
this phylogenetic hormone. This should provide the struc-
tural basis for the biological pathway of conformation–
information–transformation.
For peptide ligand–receptor interactions, there are three
general approaches that can be utilized to extract structural
information [4]: a peptide (ligand)-based approach, a
receptor-based approach, and approaches that target the
ligand–receptor complex. In many systems of biological
importance, structural characterization of the receptor, and
peptide–receptor complexes, is extremely difficult. This is
especially true for the membrane-associated G-protein
(guanine nucleotide-binding regulatory protein)-coupled
receptors, through which AII and most peptide hormones
exert their biological activity [5]. Structural determination of
these proteins has progressed slowly [6], mainly because of
technical difficulties in purifying and handling integral
membrane proteins. The instability of these proteins in
environments lacking phospholipids and the tendency for
them to aggregate and precipitate has hindered application
Correspondence to I. P. Gerothanassis, Department of Chemistry,
Section of Organic Chemistry and Biochemistry, University of
Ioannina, Ioannina GR-45110, Greece.
Fax: + 30651098799, Tel.: + 30651098397,
E-mail: ; URL: www.uoi.gr
Abbreviations: AII, angiotensin II; AT
1
, AII receptor type 1;
CSD, chemical shift deviation.

(Received 3 September 2002, revised 9 December 2002,
accepted 20 December 2002)
Eur. J. Biochem. 270, 849–860 (2003) Ó FEBS 2003 doi:10.1046/j.1432-1033.2003.03441.x
of standard structure determination techniques to these
biomolecules.
In the free ligand (peptide)-based approach, the
conformational distribution of the peptide hormone, such
as AII, in solution is investigated, on the grounds that
this parameter is involved in their binding to the
receptor. As a corollary of this argument, it has often
been assumed that the conformation of the hormone in
the complex corresponds to the predominant conforma-
tion in solution according to the key-to-lock model.
However, an alternative molecular recognition process,
the so-called ÔzipperÕ model, has been suggested in several
instances [7–11]. The key question therefore is whether
structural motifs of a flexible peptide ligand in solution
can be retained during the early stages of receptor–
peptide recognition processes [12].
AII has been extensively investigated in solution during
the last 40 years with a variety of techniques, including
theoretical, physicochemical, and spectroscopic. The results
have been interpreted in terms of various models such as
an a-helix [13], b-turn [14–17], cross-b-forms II [18],
antiparallel pleated sheet [19,20], c-turn [19], random coil
[21,22], inverse c-turn [23], side chain ring cluster [24], and
other structures [25–29]. It is evident that several of the
reported models are not consistent with each other and
that there is no general consensus on the solution
conformation of AII.

Here we present detailed high-field 2D
1
H-
1
HTOCSY
and 2D
1
H-
1
H NOESY NMR studies of AII, at low
temperature ( 277 K) in aqueous solution, at neutral pH
( 5.7). The high-resolution structures, calculated from
NMR-derived restraints, provide the first experimental
evidence that the hormone adopts a U-shaped (VIb turn)
folded structure. The conformational features of AII in the
solution state (representing the free ligand conformation)
were compared with the conformation of the hormone
complexed to the high-affinity mAb Fab131 (representing
the receptor-bound conformation) as determined by X-ray
crystallography [30]. This antibody has the unusual
property that it was not generated against AII, but rather
against an anti-idiotypic Ig with a mAb to AII, which
renders this antibody an anti-(anti-idiotypic) Ig. The high
affinity for AII of the original mAb was passed on to
mAb131 through structural determinants on the anti-
idiotypic Ig.
Materials and methods
Peptide synthesis and sample preparation
AII was synthesized according to a standard stepwise solid-
phase procedure using Fmoc/tBu chemistry [31,32]. Peptide

purity was assessed by analytical HPLC (Nucleosil-120 C
18
;
reversed phase; 250 · 4.0 mm), mass spectrometry
(FABMS, ESIMS), and amino-acid analysis. The samples
were prepared for NMR spectroscopy by dissolving the
peptide in 0.01
M
potassium phosphate buffer (pH 5.7),
containing 0.02
M
KCl. 2,2-Dimethyl-2-silapentanesulfo-
nate (DSS) was added to a concentration of 1 m
M
as an
internal chemical shift reference. Peptide concentration was
commonly 4 m
M
in 90%
1
H
2
O/10%
2
H
2
O. Trace amounts
of NaN
3
were added as a preservative.

NMR spectroscopy: determination of
distance restraints
Preliminary NMR spectra were acquired at 400 MHz
using a Bruker AMX-400 spectrometer in the NMR
Center of the University of Ioannina. High-field NMR
spectra were acquired at 750 MHz using a Varian Unity
750 spectrometer at 277 K in the European Large Scale
Facility at Utrecht University. The WATERGATE pulse
sequence [33] was used for solvent suppression. All proton
2D spectra were acquired using the States-TPPI method
for quadrature detection, with 2K · 512 complex data
points and 16 scans per increment for 2D TOCSY and 64
scans for 2D NOESY experiments, respectively. The
mixing time for TOCSY spectra was 80 ms. Mixing times
for NOESY experiments were set to 100, 200, 350 and
400 ms to determine NOE build up rates. A mixing time of
350 ms provided sufficient cross-peak intensity without
introducing spin-diffusion effects in the 2D NOESY.
Phase-sensitive 2D NOESY was used for specific assign-
ment and for estimation of proton–proton distance con-
strains. Data were zero filled in t
1
to give 2K · 2K real
data points, and 90° phase-shifted square cosine–bell
window function was applied in both dimensions. All
spectra were processed by using NMRPipe software
package [34] and analysed with NMRVIEW [35].
Interproton distances for AII were derived by measuring
cross-peak intensities in the NOESY spectra. Intensities
were calibrated to give a set of distance constraints using the

NMRVIEW software package [35]. NOEs cross-peaks were
separated into three distance categories according to their
intensity. Strong NOEs were given an upper distance
restraint of 3.0 A
˚
, medium NOEs a value of 4.0 A
˚
,andweak
NOEs 5.5 A
˚
. The lower distance limits were set to 1.8 A
˚
.
A natural abundance
1
H-
13
C HSQC NMR spectrum was
acquired on a Bruker Avance 600 MHz spectrometer at
277 K in Utrecht. The spectrum was acquired with
2K · 400 points, with 48 scans per increment. The t
1
dimension was zero-filled to 1K, to give 1K · 1K real
points, and 90° square cosine–bell window function was
applied in both dimensions.
Structure calculations
All calculations were performed with CNS [36] using the
ARIA setup and protocols [37,38], as described by Bonvin
et al. [39]. Covalent interactions were calculated with the 5.2
version of the PARALLHDG parameter file [37,38] based

on the CSDX parameter set [40]. Nonbonded interactions
were calculated with the repel function using the PROLSQ
parameters [41] as implemented in the new PARALLHDG
parameter file. The OPLS nonbonded parameters [42] were
used for the final water refinement including full van der
Waals and electrostatic energy terms.
A simulated annealing protocol in Cartesian space was
used starting from an extended conformation, and consisted
of four stages: (a) high-temperature simulated annealing
1
stage (10 000 steps, 2000 K); (b) a first cooling phase from
2000 to 1000 K in 5000 steps; (c) a second cooling phase
from 1000 to 50 K in 2000 steps; finally (d) 200 steps of
energy minimization. The time step for the integration was
set to 0.003 ps.
850 A. G. Tzakos et al.(Eur. J. Biochem. 270) Ó FEBS 2003
The structures were subjected to a final refinement
protocol with explicit waters by solvating them with a 8 A
˚
layer of TIP3P water molecules [42]. The resulting structures
were energy minimized with 100 steps of Powell steepest
descent minimization and the structure stereochemistry was
evaluated through
PROCHECK
[43]. Restraint numbers and
structural statistics for AII are presented in Table 1.
Results and discussion
High-resolution NMR structure of AII
in aqueous solution
High-field NMR spectroscopy. The NMR experiments

were performed at low temperature (277 K) to limit the
conformational ensemble of the hormone in solution and to
avoid unfavourable correlation times (i.e. when s
c
x
0
@ 1),
which result in minimum NOE intensities. Resonance
assignments were made using standard high-field 2D
methods [44] and are given in Table S1 of the supplementary
material. The primary NMR data used in structure
calculations were interproton NOEs obtained from
1
H–
1
H
2D NOESY experiments (Fig. 1). A list of NOEs, used for
the structure calculations, is given in Table S2 of the
supplementary material.
To investigate whether an amide proton is directly
involved in intramolecular hydrogen bonding, the amide
proton temperature coefficients (Dd/DT) were measured.
Exposed NHs typically have gradients in the range of )6.0
to )8.5 p.p.b./K, hydrogen-bonded or protected NHs
apparently have Dd/DT of )2.0 to ±1.4 p.p.b.ÆK
)1
[45].
For peptide fragments, however, because of conformational
averaging Dd/DT values may lie between )28 and
+12 p.p.b.ÆK

)1
, resulting in a correlation between the
gradient and structure that lies outside the rules mentioned
above [45]. A plot of Dd/DT vs. the chemical shift deviation
(CSD) of the measured amide proton resonances at 277 K
(Fig. 2), with appropriate random coil chemical shift
correction [46–48], provides a better correlation with partial
structuring of a flexible linear peptide. The dashed line
(Dd/DT ¼ )7.8 (CSD) )4.4) represents the cut off of Dd/DT
between exposed and sequestered NHs of proteins. Gradi-
ents above the dashed line indicate exposed NHs, whereas
those below indicate sequestered NHs. As can be seen in
Fig. 2, all the backbone NH, with the exception of the Arg2,
are above the dashed line, indicating that these peptide
protons are somewhat exposed. The Arg2 backbone NH is
most probably implicated in the formation of an intramo-
lecular hydrogen bond (see discussion below). Low Dd/DT
values for the backbone NH of Arg2 have been found in
cyclic analogues of AII, suggesting shielding from the
solvent, but with no rationalization about the structural
origin of this effect [29].
1
H
a
,
13
C
a
and
13

C
b
chemical shifts are known to be
strongly dependent on the nature of protein/peptide secon-
dary structure. Figure 3 shows a region of the natural
abundance
1
H-
13
C HSQC and the secondary shifts (devi-
ation of the observed chemical shifts from the random coil
chemical shift values [46,47] per residue of the amino-acid
sequence of AII). The results are indicative, in a qualitative
way, of a folded structure for the central fragment of AII.
Interestingly, His6 illustrates the largest deviation of
13
C
a
chemical shifts from the random coil values. The origin of
this phenomenon could be due to specific secondary features
of the hormone affecting the observed chemical shifts (ring
current, electric field, hydrogen-bond effects).
13
C-NMR
resonance assignments and chemical shifts are given in
Table S3 of the supplementary material.
3
J
HN-Ha
backbone-coupling constants were also extracted

from the NMR spectra. This parameter tends to be small
(<6.0 Hz) in residues in a a-helical conformation and large
(>8.0 Hz) when extended; intermediate values (6–8 Hz) do
not allow an unambiguous structural categorization [44].
For the Val3 and Ile6 residues, the
3
J
HN-Ha
values are
slightly bigger than 8 Hz (see Table S4 of the supplementary
material), and are indicative of an extended conformation.
For the residues Arg2, Tyr4, His6 and Phe8 in AII, this
parameter is not structurally discriminatory between the two
limiting cases. The intermediate values (6.4 Hz) of the
backbone couplings for Tyr4 and His6 are possibly the
result of twisting or bending in the middle of the amino-acid
sequence of the hormone [49].
Structure calculations and analysis
The presence of conformational averaging in linear peptides
can complicate the calculation of singular structures. In
particular, the use of intraresidue and sequential NOEs,
which are likely to have substantial contributions from
folded and unfolded states, is problematic [50]. Two sets of
structure calculations were implemented, considering: (a)
sequential (|i ) j| ¼ 1), medium (1 < |i ) j| < 4) and long-
range (|i ) j| > 4) NOE cross-peaks and (b) medium
(1 < |i ) j| < 4) and long-range (|i ) j| > 4) NOE cross-
peaks. The resulting conformational ensembles for cases (a)
Table 1. Summary of input restraint and structural statistics. Based on
the 13 structures, obtained by simulated annealing in CNS followed by

refinement in explicit water using NOE distance restraints, dihedral
angle restraints, bond, angles, impropers, dihedral angle, van der waals
and electrostatic energy terms.
RMSD (A
˚
) with respect to mean
Heavy backbone atoms (residues 1–8) 0.73 ± 0.23
All heavy atoms (residues 1–8) 1.35 ± 0.29
Number of experimental restraints
Total NOEs 262
Intraresidue NOEs 87
Interresidue sequential NOEs (|i)j| ¼ 1) 114
Interresidue medium-range
NOEs (1 < |i)j| < 4)
59
Interresidue long-range NOEs (|i)j| > 4) 2
Restraint violations statistics
NOE distances with violations > 0.3 (A
˚
)0
RMSD for experimental restraints (A
˚
) 0.087 ± 0.005
CNS energies from SA
a,b
F
vdw
(kcalÆmol
)1
) 207 ± 37

F
elec
(kcalÆmol
)1
) ) 27 ± 4
a
Force constants are described in Materials and methods.
b
The
Lennard–Jones 3–7 and coulomb energy terms were calculated
within CNS using the OPLS nonbonded parameters (as described
in Materials and methods).
Ó FEBS 2003 Comparison of the free and bound structure of angiotensin II (Eur. J. Biochem. 270) 851
Fig. 1. Selected region of a 750-MHz NOESY spectrum (350 ms mixing time) of AII (90% H
2
O/10% D
2
O). Cross-peaks characteristic of the folded
conformation are annotated. The red arrows denote the presence of the minor cis isomer.
852 A. G. Tzakos et al.(Eur. J. Biochem. 270) Ó FEBS 2003
and (b) suggested the same overall fold and side chain
orientation for the hormone. In addition, we checked for the
presence of conformational averaging by performing
ensemble-average refinement with complete cross-validation
against the number of conformers [51,52]. This procedure
indicated that a single conformer was sufficient to satisfy the
experimental restraints (Figure S1 of the supplementary
material).
In the second set of calculations, 173 sequential and
medium range NOEs and two long-range NOEs were used

as distance restraints for AII (Table S2 of the supplementary
material). No explicit dihedral or hydrogen-bonding
restraints were applied. Structure calculations were per-
formed using a simulated annealing protocol, following
ARIA/CNS setup [36–40]. A family of 200 structures was
calculated. Forty-eight structures with the lowest energy and
NOE violations of no larger than 0.25 A
˚
were selected after
the final refinement in explicit water. The conformation of
the hormone does not change significantly in the final stages
of refinement and is mainly determined by the NMR data.
The quality of the structures, however, improved after water
refinement because both electrostatic and full Lennard-
Jones potentials are used. From the family of the 48
structures, 13 structures were selected having the best
allowed regions in the Ramachandran plot (Fig. 4). The
RMSD value of the ensemble of the 13 calculated structures,
with respect to mean structure, for backbone atoms
(residues 1–8) was found to be 0.73 ± 0.23 A
˚
and for all
heavy atoms 1.35 ± 0.29 A
˚
(Table 1).
The most well-defined fragment of the peptide hormone
contains the amino-acid residues 3–7. Figure 5A (a,b)
provides a superposition of backbone and heavy atom of
the entire ensemble of the 13 calculated structures for the
3–7 fragment. Figure 5B (a) illustrates a representative

conformer whose structure is the closest to the average
co-ordinates of the ensemble.
The structures calculated from NMR-derived restraints
have a well-defined U-shaped conformation for the back-
bone with a trans His-Pro amide bond. The RMSD values
for the backbone C
a
,N,C¢ atoms from the mean structure
for the 3–7 fragment is 0.14 ± 0.05 A
˚
and for all the heavy
atoms (C
a
,N,C¢, O) the RMSD is 0.32 ± 0.15 A
˚
.The
RMSD values of the backbone atoms from the mean value
for the 4–7 fragment is 0.10 ± 0.04 A
˚
and for all heavy
atoms the RMSD is 0.32 ± 0.16 A
˚
. The N-terminal and
C-terminal tails are less well defined by the available
restraints.
The present NMR study clearly demonstrates that in
aqueous solution, even small peptide hormones can adopt
favoured, rather well defined conformations. Thus, in
aqueous solution, AII adopts a fairly compact structure
with its C-termini and N-termini approaching to within

 7.2 A
˚
of each other. Furthermore, the side chains of Arg2,
Tyr4, Ile5, His6 are oriented on one side of a plane defined
by the peptide backbone, and the Val3 and Pro7 are pointed
to opposite directions. The Tyr4 side chain is oriented inside
the folded conformation of the molecule, and Arg2 is
exposed to the solvent, as illustrated in Fig. 5B,a. The
stabilization of the folded conformation can be explained by
the stacking of the Val3 side chain with the Pro7 ring (this is
consistent with the observed long-range NOE cross-peaks
and the distance between, e.g. Val3 C
c
and Pro7 C
c
which is
Fig. 3. (A) Selected region of a
1
H-
13
CHSQCspectrumofAIIand(B)
secondary chemical shifts (deviation of the observed chemical shifts from
the random coil chemical shift values) per residue of the amino-acid
sequence of AII.
Fig. 2. NH Dd/DT vs. CSD for AII in water solution and pH 5.7. The
dashed line corresponds to Dd/DT ¼ )7.8 (CSD) )4.4, which provides
the optimum differentiation of sequestered NHs in the protein data-
base.
Ó FEBS 2003 Comparison of the free and bound structure of angiotensin II (Eur. J. Biochem. 270) 853
 3.2 A

˚
) and by a hydrophobic cluster formed by the Tyr4,
Ile5 and His6 side chains (Fig. 6). Analysis of the distance
between the N-terminus and C-terminus of the peptide for
the individual conformers indicates that the compact
conformation of the octapeptide is not stabilized by a salt
bridge between the two charged groups.
It is interesting to compare the overall fold of AII from
the present study with previously reported conformations
for AII in solution. An Ôopen turnÕ conformation for the
Tyr4-Ile5 residues has been observed with NMR by
Nikiforovich et al. [29] in cyclic AII analogues, containing
a disulfide bridge between positions 3 and 5. Detailed
conformation–biological activity studies of Fermandjian
et al. [53] in a series of AII analogues substituted in Ile5,
confirmed the steric influence of residue 5 on the organiza-
tion of Tyr4 and His6 side chains. In addition, structure–
activity relationship studies highlighted the requirement of a
lipophilic and b-branched hydrocarbon moiety in the fifth
position for high pressor activity of AII analogues [54]. In
accordance with our model, this could be explained through
the formation of a hydrophobic cluster by the Tyr4, Ile5 and
His6 side chains, as illustrated in Fig. 6B.
Recently, NMR studies of AII in a phospholipid micelle
solution were interpreted in terms of a well-defined hairpin
compact structure, similar to our aqueous solution struc-
ture, with the N-terminus and C-terminus approaching to
within 7.6 A
˚
of each other [23] and an inverse c-turn

encompassing residues His6, Pro7 and Phe8. The Tyr4, His6
and Phe8 side chains were found to be close together in
space. The orientation of the Tyr4 side chain is similar in the
two structures, with a dihedral (v
1
) angle 65(8)° in the
phospholipid environment and 46.5 (5.1)° in aqueous
solution. The Asp1 side chain carboxylic group was
suggested to be involved in hydrogen-bonding interaction
with either the N-terminal amino group or the Arg2 side
chain amino group, whereas in our studies a hydrogen bond
is formed with the backbone NH of Arg2. Similar
investigations in conformationally restrictive environment
(DodChoP micelles) [15], by the use of ultraviolet resonance
Raman and absorption studies, provided evidence that AII
adopts a folded turn-like structure and that Tyr4 is either
involved in a hydrogen bond through its hydroxy group or
it is buried in a hydrophobic milieu. The latter seems to be a
more plausible explanation in the frame of the observed
hydrophobic cluster in our study.
In conclusion, the similarity of the folded structure of AII
in aqueous solution and in conformationally restrictive
phospholipid environment clearly demonstrates that the
presence of a hydrophobic–hydrophilic interface does not
play a determinative role in conferring the structure of AII.
X-ray structure of AII bound to the mAb Fab131:
comparison with the NMR solution structure
The phenomenon of conformational stabilization [55] and
selection between different antigen conformers has been
demonstrated by means of antibodies that act on a

population of antigen molecules with different extents of
conformational order [56,57]. Thus, a Ôsurrogate systemÕ
that consists of a high-affinity monoclonal antibody
(mAb131) and AII had been used to study a bound
conformation of AII [30]. The 3D structure of the AII–Fab
complex has been refined by Garcia et al. [30]. The binding
site of the antibody is very deep and narrow. This has two
effects: (a) it markedly increases the exposed surface area of
the free mAb; (b) it creates space from which it is easy to
Fig. 4. Ensemble Ramachandran plots of the 13 solution structures of AII.
854 A. G. Tzakos et al.(Eur. J. Biochem. 270) Ó FEBS 2003
exclude solvent by filling the cavity. The most buried
residues of AII are of the central AII sequence Tyr4-Ile5-
His6-Pro7, which is also the most immunogenic epitope of
the peptide [58–60]. Most substitutions of these immuno-
genic residues abolish binding to both mAb131 and AT
1
.
The AII bound to the mAb adopts a compact confor-
mation with two turns (Fig. 5B,b). The first turn involves
residues Asp1 and Arg2 and brings the N-terminus of the
peptide in spatial proximity to the C-terminus of the
peptide. It was suggested that the stabilization of such a
tight conformation may result from the formation of a salt
bridge between the termini and a hydrogen bond between
the –NH
3
+
terminal of Asp1 and the main chain carbonyl
group of Ile5 [30]. The second turn involves the residues Ile5,

His6, Pro7, and the centre of this turn is lodged in the
deepest region of the binding site. This tight VIb-type turn
contains a cis His6-Pro7 peptide bond (x  40
°
)which
results in a 90° twist of this part of the backbone with
respect to the rest of the molecule. Evidence for the presence
of a highly populated VIb turn-like conformation was also
Fig. 5. Structure of AII. (A) Solution conformation of AII. (a) The 13 structures calculated for AII overlaid using the N, C
a
and C¢ atoms of residues
3–7. (b) Superposition of the backbone and heavy atoms of the fragment 3–7 of AII. (B) Comparison of a representative conformer of AII with
structure closest to the average co-ordinates (the blue colour denotes the side chains of Arg2, Tyr4, Ile5, His6 and the yellow the side chains of Val3
and Pro7) (a) with the the X-ray structure of AII in the Fab131–AII complex [30]
3
(b).
Ó FEBS 2003 Comparison of the free and bound structure of angiotensin II (Eur. J. Biochem. 270) 855
provided for the cis X-Pro isomers of several peptides with
the sequence motif X-Pro-Phe [61] (X stands for aromatic
amino acid). This specific conformational feature may be of
importance for the conformation of the AII complexed to
the AT
1
G-protein-coupled receptor and the activation of
the receptor. Interestingly, a cis to trans conformational
switch isomerization of the 11-cis-retinal chromophore of
rhodopsin is of primary importance for stimulation of the
receptor and transformation to the signalling state [62].
2
The present NMR data provide the basis for a quantitative

comparison of the structure of AII in solution with the X-ray
structure of AII complexed to the Fab131 mAb. In Fig. 7A, a
sequence alignment is represented of the backbone of the
conformational ensemble of AII in solution state with the
backbone of the X-ray structure. In Fig. 7B, the models are
superimposed using structural alignments only. Table 2
illustrates the RMSD values obtained after the sequence and
structure alignment of the two structures by using the
program Profit1.8. Remarkably, the superposition of the
solution state and the bound structure of AII exhibits small
RMSD positional differences between the two structures.
Fig. 7. (A) Sequence alignment of the fragment 4–7 of the backbone of
the 13 ensemble solution structures of AII (brown colour) superimposed
on the X-ray structure (dark blue colour) and (B) structure alignment of
the fragment 4–7 of the 13 ensemble structures of AII to the fragment 3–6
of the X-ray structure of AII.
Fig. 6. Structure of a representative folded conformer of AII showing the van der Waals contacts between the side-chains of residues Val3 and Pro7 (A)
and Tyr4, Ile5, and His6 (B). Carbon atoms are shown in grey, oxygen atoms in red, nitrogen atoms in blue, and hydrogen atoms in white.
Table 2. Average backbone atomic root mean square positional differ-
ences between the X-ray structure of AII and the ensemble of 13 AII
calculated structures. Superposition is based on sequence and structure
alignment.
Residue number
range in the X-ray
structure of AII
Residue number
range in the solution
average structure of AII
Backbone
(C¢,N,C

a
)
RMSD (A
˚
)
2–7 2–7 1.99 ± 0.04
3–7 3–7 1.90 ± 0.04
3–6 3–6 1.30 ± 0.03
4–7 4–7 1.27 ± 0.02
2–5 3–6 0.82 ± 0.03
2–6 3–7 1.07 ± 0.05
3–6 4–7 0.76 ± 0.03
856 A. G. Tzakos et al.(Eur. J. Biochem. 270) Ó FEBS 2003
Thus, it can be concluded that small rearrangements of the
backbone on binding are required by a mean value of about
1.27 ± 0.02 A
˚
for sequence alignment and 0.76 ± 0.03 A
˚
for structure alignment of the most immunogenic epitope 4–7
of AII. This part of the peptide hormone therefore is quite
rigid and prearranged in the solution state for binding at the
receptor–antigen recognition site.
The common features among the solution structure of
AII and the bound conformation to the antibody Fab131
are:
(a) the first turn (Asp1 and Arg2) which induces the
orientation of the N-terminal part to the C-terminal
part of the molecule;
(b) the second turn (His6, Pro7, Phe8);

(c) the third turn (Ile5, His6 and Pro7).
The crystallographic distance of the Asp1 side chain
(OD1) to the main chain NH of the proceeding Arg2 is
 2.9 A
˚
, underlying the possibility of the formation of a
side chain–main chain hydrogen bond. This distance is
comparable to that obtained from the NMR structure in
solution ( 2.8 A
˚
) and consistent with the measured NH
temperature coefficients, which revealed the possible
involvement of the Arg2 backbone NH proton in intra-
molecular hydrogen bonding. The experimental data of
this study therefore demonstrate the formation of an Asx-
like turn with the side chain carbonyl group of aspartate
hydrogen-bonded to the main chain NH of the preceding
arginine, forming a stable heptamer ring, both in the X-ray
structure and the solution structure of AII (Fig. 8). Indeed,
aspartate residues at the N-termini of short polypeptides
have shown a stabilizing influence [63,64]. These effects are
thought to result from an interaction of the negative charge
with the dipole of the polypeptide chain. Examination by
Wan and Milner-White [65] of several high-resolution
crystal structures of the ways that side chain carboxylates
form hydrogen bonds with main chain atoms revealed a
high incidence of Asx motifs with the aspartate or
asparagine as the first residue. Specifically Asx motifs
occur with the side chain aspartate carboxyl group
hydrogen-bonded to a main chain NH group of the

residue two to three amino acids ahead.
Several structure–activity studies of AII have delineated
the requirements for agonist activity, highlighting Tyr4 and
Phe8 as basic requirements for high pressor activity of AII;
substitution with other amino acids results in antagonistic
analogues. Furthermore, Tyr4 has been suggested to be a
switch residue responsible for receptor activation. Specific-
ally, it has been proposed that the activation of the AT
1
receptor from the basal state requires a key interaction
between Asn111, in the transmembrane helix III (TM3) of
the receptor, and the Tyr4 of AII [66,67]. Interestingly, the
side chain of Tyr4 of the free hormone in aqueous solution
adopts a very low RMSD value, and it is oriented inside the
overall fold of the molecule, whereas in its bound state it is
oriented outside the fold towards the receptor site. Very
probably, this  130° rearrangement in the v
1
angle requires
a small energy conformational barrier for the initial step of
the receptor–peptide recognition and could be responsible
for initiating a biochemical cascade upon the interaction of
Tyr4 with Asn111 [68].
Constructively, the overall fold of AII in aqueous
solution state is reminiscent of the conformation observed
when AII is bound to the mAb Fab131. However, in the
crystal structure of the complex, a hydrogen bond between
the -NH
3
+

terminal of Asp1 and the main chain carbonyl
group of Ile5 is observed, which is not the case in aqueous
solution. In the X-ray structure, AII has a cis His-Pro amide
bond. In our NMR spectra of AII in solution, we do
observe additional peaks that can be attributed to a minor
population of less than 10% with the His-Pro bond in the cis
conformation (Fig. 1). The presence of such a conformation
in the crystal complex (with x  40
°
) and its low fraction in
aqueous solution indicate that it is energetically strained,
and that the extensive intermolecular interactions observed
in the complex are necessary to compensate for the free
strain. For example, there are several close contacts of His6
and Pro7 of AII to Ser
L91
,Tyr
L95
,Arg
H52
and Tyr
L92
,
Ala
H33
,Arg
H52
and Arg
H99
, respectively, in the Fab131

ÔreceptorÕ site [30].
The results obtained in our studies suggest that the folded
conformation of AII in aqueous solution is optimal for
receptor–antigen recognition, and that binding and the
energetic cost of deforming it into the bound conformation
is compensated by the energetic benefit that could be
obtained from intermolecular contacts in the bound state.
We argue therefore that pre-existing subpopulations of
ligand–peptide conformers preferentially bind to their
corresponding receptor in a frame of ÔcomplementarityÕ.
As pointed out by Porschke and Eigen [69], a mechanism
for information transfer must satisfy the dual criteria of
selectivity and speed. High selectivity is most rapidly
achieved by having a relatively large recognition site
(pointing out common structural features among free and
bound ligand). Conformational studies therefore of small
Fig. 8. The Asx-like motif of AII in the NMR structure (brown colour)
and in the X-ray structure (blue colour). The hydrogen bond is shown by
the dashed line.
Ó FEBS 2003 Comparison of the free and bound structure of angiotensin II (Eur. J. Biochem. 270) 857
peptide hormones, such as AII, in aqueous solution may
have important consequences in delineating structure–
function relationships and the principles of biomolecular
hormone–receptor interaction and recognition. Further
work along these lines is currently underway in our
laboratories.
Acknowledgements
A.G.T. acknowledges the Federation of EuropeanBiochemical Societies
(FEBS) for a summer fellowship. The 750 and 600 MHz spectra were
recorded at the SONNMR Large Scale Facility in Utrecht, which is

funded by the ÔAccess to Research Infrastructures Programm of the
European UnionÕ (HPR1-CT-1999-00005). We also thank the SON-
NMR Large Scale Facility for the use of the computational facilities.
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Supplementary material
The following material is available from http://
www.blackwellpublishing.com/products/journals/suppmat/
EJB/EJB3441/EJB3441sm.htm
Table S1. Resonance assignments made using standard
high-field 2D methods.
Table S2. List of NOEs.
Table S3.
13
C-NMR resonance assignments and chemical
shifts.
Table S4.
3
J
HN-Ha

values.
Fig. S1. Ensemble-averaging cross-validation results
against the number of conformers in the ensemble.
860 A. G. Tzakos et al.(Eur. J. Biochem. 270) Ó FEBS 2003